PROJECT SUMMARY Ferroptosis is a form of regulated cell death that generates reactive oxygen species (ROS) in an iron- dependent fashion. The most important of the ROS are lipid peroxides, which self-propogate along the plasma membrane and result in the accumulation of oxidatively damaged lipids. These lipid peroxides lead to a loss of membrane integrity, leakage of cellular and subcellular content and, ultimately, cell death. The main protective mechanism against ferroptosis relies on a gluathione-dependent peroxidase (GPX4); this enzyme converts lipid peroxides to lipid alcohols, thus blocking cell death. Defects in this protective pathway have been implicated in the etiology of a number of neurodegenerative disorders. Conversely, abrogation of GPX4 has sensitized drug-resistant cancer cells to chemotherapeutics. Unfortunately, our understanding of the mechanisms underlying ferroptosis remain limited, but one possibility of ferroptotic regulation may be found in the enzyme acyl-CoA synthetase long-chain 4 (ACSL4). This enzyme converts free long-chain fatty acids into fatty acyl-CoA esters, the building blocks of the phospholipids that are fated to propagate ferroptosis. Knockout of ACSL4 has been shown to stave off ferroptosis in multiple in vivo and in vitro models. Interestingly, the ACSL4 gene can produce two isoenzymes via alternative splicing; these isoforms differ in length and in localization, where the long form co-localizes to lipid droplets (LDs) and the short form co- localizes to the plasma membrane (PM). We hypothesize that this differential localization modulates ferroptotic sensitivity as the sequestration of lipid peroxides to the LDs may prevent death by ferroptosis. In order to address this question, cell lines will be created in which endogenous ACSL4 has been eliminated and replaced with a different expression construct for the two major splice forms of ACSL4. Moreover, these splice forms will be targeted to various subcellular localizations to investigate the role of these compartments in the execution of ferroptotic cell death. Additionally, since ACSL4 is integral to lipid metabolism, we will investigate the composition of fatty acids via Raman spectroscopy and characterize fatty acid flux through lipidomics. This project addresses an important step in the regulation of ferroptosis, namely how sequestration of damaged lipids away from critical membrane regions may negatively affect ferroptotic cell death. The enzyme that governs this sequestration, ACSL4, thus has great pharmacologic potential, not only as a drug target for novel anti-cancer drugs, but also in age-related neurological diseases.